Regulation at the stage of alternative RNA splicing is common in the expression of most genes in mammallian cells. Different isoforms of many proteins are expressed in cells after activation of the Ras signaling pathway. However, the mechanisms controlling the underlying alternative RNA splicing processes have not been well studied. Inclusion of variant exons of the cell surface protein CD44 is controlled by Ras activation. This gene contains 10 constitutive exons with a string of 10 variable exons between constitutive exons 5 and 6. CD44 is expressed at high levels in normal stem cells, most cancer stem cells, and in mesenchymal cells after the epithelial to mesenchymal transition. Furthermore, CD44 expression promotes cell migration and metastatic spread of tumors. These latter activities are dependent upon alternative splicing of CD44 which produces isoforms containing v6 and other exons. These variant isoforms of CD44 also serve as co-receptors on the cell surface and, in the presence of the appropriate ligand, activate Ras signaling. Thus, the isoforms of CD44 are part of a positive feedback loop sustaining their own expression. We propose a loss-of-function screen using libraries of pooled shRNA retroviral vectors to identify genes important for the alternative splicing of CD44 after activation of the Ras pathway. We are particularly interested in genes that: encode cell surface proteins, serve as co- receptors with isoforms of CD44, and encode nuclear factors controlling alternative splicing downstream of Ras activation. An initial screen with a modest number of shRNA vectors has been completed and candidate genes, identified through shRNA silencing, are being validated. The criteria for this validation are described. Two of these candidate genes are interesting, angiogenin (ANG1), important in angiogenesis and structurally a member of the ribonuclease family, and SRp54, an RNA splicing factor known to be important for inclusion of exon 10 in the tau protein. A second screen using a larger pooled library of shRNA lentivirus vectors has been initiated. During this screen, enrichments of specific hairpins in selected pools will be assayed by microarrays. We have recently reported that many genes with tandem polyadenylation sites are expressed with short 3'UTRs in proliferative cells versus long 3'UTRs in quiescent cells. The long 3'UTRs contain at least twice the number of conserved target sites for regulation by microRNAs as the short 3'UTRs. This suggests that gene regulation through recognition of conserved sites in the 3'UTR might be more extensive in quiescent cells than proliferative cells. We propose to test this hypothesis by quantitatively determining the effect of recognition by microRNA and RNA binding proteins on gene expression in quiescent cells. PUBLIC HEALTH RELEVANCE: Alternative RNA splicing is a central aspect of most normal and disease processes. Little is known about the factors and pathways that regulate alternative RNA splicing. We propose to study how alternative splicing of CD44 is regulated by the Ras signaling pathway. These studies will both increase our knowledge of mechanisms controlling alternative splicing and as well the factors that control CD44 activity in diseases such as cancer.